Keratohyalin-like granules in lizard epidermis: evidence from cytochemical, autoradiographic, and microanalytic studies

Epidermal sloughing in lizards is determined by the formation of an intraepithelial shedding complex in which keratohyalin-like granules are formed. The chemical nature of these granules is unknown, as is their role in keratinization. The goal of this study was to test whether they contain some amino acids similar to those found in mammalian keratohyalin. The embryonic and regenerating epidermis of lizards are useful systems to study the formation of these granules. Histochemically keratohyalin-like granules react to histidine and contain some sulfhydryl groups (cysteine). X-ray microanalysis shows that these granules contain sulfur and often phosphorus, two elements also present in the mature clear, oberhautchen, and beta layer. Instead the mesos, alpha, and lacunar layers contain only sulfur. Most sulfur is probably in a disulfide-bonded form, particularly in mature cells of the shedding complex, in large keratohyalin-like granules, and in the beta-keratin layer. Early differentiating beta-keratin cells have the maximal incorporation of tritiated proline, whereas tritiated arginine is slightly more concentrated in the basal layer of the epidermis. A high uptake of tritiated histidine is observed mainly in keratohyalin-like granules of the clear layer, but also in the oberhautchen layer and forming the alpha-lacunar layer. Immunogold electron microscopy shows that keratohyalin-like granules do not localize keratin but are embedded within a keratin network. These results suggest that keratohyalin-like granules of lizards, like mammalian keratohyalin, contain some sulfur-rich and histidine-rich proteins. These granules participate in the process of hardening of the clear layer that molds the spinulae of the deeper oberhautchen to form the superficial microornamentation.     

Synthesis of interkeratin matrix in differentiating lizard epidermis: an ultrastructural autoradiographic study after injection of tritiated proline and histidine

During epidermal differentiation in mammals, keratins and keratin-associated matrix proteins rich in histidine are synthesized to produce a corneous layer. Little is known about interkeratin proteins in nonmammalian vertebrates, especially in reptiles. Using ultrastructural autoradiography after injection of tritiated proline or histidine, the cytological process of synthesis of beta-keratin and interkeratin material was studied during differentiation of the epidermis of lizards. Proline is mainly incorporated in newly synthesized beta-keratin in beta-cells, and less in oberhautchen cells. Labeling is mainly seen among ribosomes within 30 min postinjection and appears in beta-keratin packets or long filaments 1-3 h later. Beta-keratin appears as an electron-pale matrix material that completely replaces alpha-keratin filaments in cells of the beta-layer. Tritiated histidine is mainly incorporated into keratohyalin-like granules of the clear layer, in dense keratin bundles of the oberhautchen layer, and also in dense keratin filaments of the alpha and lacunar layer. The detailed ultrastructural study shows that histidine-labeling is localized over a dense amorphous material associated with keratin filaments or in keratohyalin-like granules. Large keratohyalin-like granules take up labeled material at 5-22 h postinjection of tritiated histidine. This suggests that histidine is utilized for the synthesis of keratins and keratin-associated matrix material in alpha-keratinizing cells and in oberhautchen cells. As oberhautchen cells fuse with subjacent beta-cells to form a syncytium, two changes occur : incorporation of tritiated histidine, but uptake of proline increases. The incorporation of tritiated histidine in oberhautchen cells lowers after merging with cells of the beta-layer, whereas instead proline uptake increases. In beta-cells histidine-labeling is lower and randomly distributed over the cytoplasm and beta-keratin filaments. Thus, change in histidine uptake somehow indicates the transition from alpha- to beta-keratogenesis. This study indicates that a functional stratum corneum in the epidermis of amniotes originates only after the association of matrix and corneous cell envelope proteins with the original keratin scaffold of keratinocytes.     

Histidine uptake in the epidermis of lizards and snakes in relation to the formation of the shedding complex

Mammalian epidermis utilizes histidine-rich proteins (filaggrins) to aggregate keratin filaments and form the stratum corneum. Little is known about the involvement of histidine-rich proteins during reptilian keratinization. The formation of the shedding complex in the epidermis of snakes and lizards, made of the clear and the oberhautchen layers, determines the cyclical epidermal sloughing. Differently from snakes, keratohyalin-like granules are present in the clear layer of lizards. The uptake of tritiated histidine into the epidermis of two lizards and one snake has been studied by autoradiography in sections at progressive post-injection periods. At 40 min and 1 hr post-injection keratohyalin-like granules were not or poorly labeled. At 3-22 hr post-injection most of the labeling was present over suprabasal cells destined to form the shedding complex, in keratohyalin-like granules of the clear layer, and in the forming a-layer but was low in the forming b-layer, and in superficial keratinized layers. The analysis of the shedding complex in the pad lamellae (a specialized scale used for climbing) of a gecko showed that the setae and the cytoplasm of clear cells among them are main sites of histidine uptake at 4 hr post-injection. In the snake most of the labeling at 4 hr post-injection was localized in the shedding complex along the boundary between the clear and oberhautchen layers. The present study suggests that, in the epidermis of lepidosaurian reptiles, the synthesis of a histidine-rich protein is involved in the formation of the shedding layer and, as in mammals, in a-keratinization.     

Ultrastructure of the embryonic snake skin and putative role of histidine in the differentiation of the shedding complex.

The morphogenesis and ultrastructure of the epidermis of snake embryos were studied at progressive stages of development through hatching to determine the time and modality of differentiation of the shedding complex. Scales form as symmetric epidermal bumps that become slanted and eventually very overlapped. During the asymmetrization of the bumps, the basal cells of the forming outer surface of the scale become columnar, as in an epidermal placode, and accumulate glycogen. Small dermal condensations are sometimes seen and probably represent primordia of the axial dense dermis of the growing tip of scales. Deep, dense, and superficial loose dermal regions are formed when the epidermis is bilayered (periderm and basal epidermis) and undifferentiated. Glycogen and lipids decrease from basal cells to differentiating suprabasal cells. On the outer scale surface, beneath the peridermis, a layer containing dense granules and sparse 25-30-nm thick coarse filaments is formed. The underlying clear layer does not contain keratohyalin-like granules but has a rich cytoskeleton of intermediate filaments. Small denticles are formed and they interdigitate with the oberhautchen spinulae formed underneath. On the inner scale surface the clear layer contains dense granules, coarse filaments, and does not form denticles with the aspinulated oberhautchen. On the inner side surface the oberhautchen only forms occasional spinulae. The sloughing of the periderm and embryonic epidermis takes place in ovo 5-6 days before hatching. There follow beta-, mesos-, and alpha-layers, not yet mature before hatching. No resting period is present but a new generation is immediately produced so that at 6-10 h posthatching an inner generation and a new shedding complex are forming beneath the outer generation. The first shedding complex differentiates 10-11 days before hatching. In hatchlings 6-10 h old, tritiated histidine is taken up in the epidermis 4 h after injection and is found mainly in the shedding complex, especially in the apposed membranes of the clear layer and oberhautchen cells. This indicates that a histidine-rich protein is produced in preparation for shedding, as previously seen in lizard epidermis. The second shedding (first posthatching) takes place at 7-9 days posthatching. It is suggested that the shedding complex in lepidosaurian reptiles has evolved after the production of a histidine-rich protein and of a beta-keratin layer beneath the former alpha-layer.     

Epidermal differentiation during ontogeny and after hatching in the snake Liasis fuscus (Pythonidae,Serpentes, Reptilia), with emphasis on the formation of the shedding complex

Differentiation and localization of keratin in the epidermis during embryonic development and up to 3 months posthatching in the Australian water python, Liasis fuscus, was studied by ultrastructural and immunocytochemical methods. Scales arise from dome-like folds in the skin that produce tightly imbricating scales. The dermis of these scales is completely differentiated before any epidermal differentiation begins, with a loose dermis made of mesenchymal cells beneath the differentiating outer scale surface. At this stage (33) the embryo is still unpigmented and two layers of suprabasal cells contain abundant glycogen. At Stage 34 (beginning of pigmentation) the first layers of cells beneath the bilayered periderm (presumptive clear and oberhautchen layers) have not yet formed a shedding complex, within which prehatching shedding takes place. At Stage 35 the shedding complex, consisting of the clear and oberhautchen layers, is discernible. The clear layer contains a fine fibrous network that faces the underlying oberhautchen, where the spinulae initially contain a core of fibrous material and small beta-keratin packets. Differentiation continues at Stage 36 when the beta-layer forms and beta-keratin packets are deposited both on the fibrous core of the oberhautchen and within beta-cells. Mesos cells are produced from the germinal layer but remain undifferentiated. At Stage 37, before hatching, the beta-layer is compact, the mesos layer contains mesos granules, and cells of the alpha-layer are present but are not yet keratinized. They are still only partially differentiated a few hours after hatching, when a new shedding complex is forming underneath. Using antibodies against chick scale beta-keratin resolved at high magnification with immunofluorescent or immunogold conjugates, we offer the first molecular confirmation that in snakes only the oberhautchen component of the shedding complex and the underlying beta cells contain beta-keratin. Initially, there is little immunoreactivity in the small beta-packets of the oberhautchen, but it increases after fusion with the underlying cells to produce the syncytial beta layer. The beta-keratin packets coalesce with the tonofilaments, including those attached to desmosomes, which rapidly disappear in both oberhautchen and beta-cells as differentiation progresses. The labeling is low to absent in forming mesos-cells beneath the beta-layer. This study further supports the hypothesis that the shedding complex in lepidosaurian reptiles evolved after there was a segregation between alpha-keratogenic cells from beta-keratogenic cells during epidermal renewal.     

Distribution and characterization of proteins associated with cornification in the epidermis of gecko lizard

The distribution and molecular weight of epidermal proteins of gecko lizards have been studied by ultrastructural, autoradiographic, and immunological methods. Setae of the climbing digital pads are cross-reactive to antibodies directed against a chick scutate scale beta-keratin but not against feather beta-keratin. Cross-reactivity for mammalian loricrin, sciellin, filaggrin, and transglutaminase are present in alpha-keratogenic layers of gecko epidermis. Alpha-keratins have a molecular weight in the range 40-58 kDa. Loricrin cross-reactive bands have molecular weights of 42, 50, and 58 kDa. Bands for filaggrin-like protein are found at 35 and 42 kDa, bands for sciellin are found at 40-45 and 50-55 kDa, and bands for transglutaminase are seen at 48-50 and 60 kDa. The specific role of these proteins remains to be elucidated. After injection of tritiated histidine, the tracer is incorporated into keratin and in setae. Tritiated proline labels the developing setae of the oberhautchen and beta layers, and proline-labeled proteins (beta-keratins) of 10-14, 16-18, 22-24 and 32-35 kDa are extracted from the epidermis. In whole epidermal extract (that includes the epidermis with corneous layer and the setae of digital pads), beta-keratins of low-molecular weight (10, 14-16, and 18-19 kDa) are prevalent over those at higher molecular weight (34 and 38 kDa). In contrast, in shed epidermis of body scales (made of corneous layer only while setae were not collected), higher molecular weight beta-keratins are present (25-27 and 30-34 kDa). This suggests that a proportion of the small beta-keratins present in the epidermis of geckos derive from the differentiating beta layer of scales and from the setae of digital pads. Neither small nor large beta-keratins of gecko epidermis cross-react with an antibody specifically directed against the feather beta-keratin of 10-12 kDa. This result shows that the 10 and 14-16 kDa beta-keratins of gecko (lepidosaurian) have a different composition than the 10-12 kDa beta-keratin of feather (archosaurian). It is suggested that the smaller beta-keratins in both lineages of sauropsids were selected during evolution in order to build elongated bundles of keratin filaments to make elongated cells. Larger beta-keratins in reptilian scales produce keratin aggregations with no orientation, used for mechanical protection.     

Ultrastructural autoradiographic and immunocytochemical analysis of setae formation and keratinization in the digital pads of the gecko Hemidactylus turcicus (Gekkonidae,Reptilia)

The modified subdigital scales of some lizards allow them to climb vertical surfaces. This is due to the action of millions of tiny setae present in the digital pads. Setae are mainly composed of beta-keratin which may have some modality of aggregation similar to that of barbs and barbules of feathers. Keratins and associated proteins are involved in the organization of setae. The formation of setae in the climbing pad lamellae of the gecko Hemidactylus turcicus has been analyzed under the electron microscope after injection of tritiated histidine and immunocytochemistry for a chick scale beta-keratin. Setae are made up of dense and pale filaments, both oriented along the longer axis of setae. Beta-keratin is present in the oberhautchen layer and in the growing setae which are highly modified oberhautchen cells. Most of the immunolabeling concentrated in the central part of setae. This cross-reactivity suggests that some epitopes in chick beta-keratin are also present in gecko setae. Four hours after injection of tritiated histidine, the labeling is localized over setae, in particular in the dense filaments and less in the pale filaments. Some labeling is also seen in the keratinaceous material present in the cytoplasm of clear cells, which are believed to mold setae. The present observations suggest that both beta-keratin and denser matrix proteins, possibly incorporating histidine, are packed into growing setae. These proteins may be mixed to form pale and dense filaments oriented along the longer axis of setae, a pattern resembling that of barb and barbule cells of feathers. The role of matrix material in the orientation of the deposited beta-keratin during setal outgrowth is discussed with the problem of barb and barbule differentiation in avian feathers.     

Immuno-cross reactivity of transglutaminase and cornification marker proteins in the epidermis of vertebrates suggests common processes of soft cornification across species

In differentiating mammalian keratinocytes proteins are linked to the plasma membrane by epidermal transglutaminases through N-epsilon-(gamma-glutamyl)-lysine isopeptide bonds to form the cornified cell envelope. The presence of transglutaminases and their protein substrates in the epidermis of nonmammalian vertebrates is not known. The present study analyses the presence and localization of the above proteins in the epidermis using immuno-cross reactivity across different classes of amniotes. After immunoblotting, some protein bands appear labelled for loricrin, sciellin, and transglutaminase in most species. These proteins are scarce to absent in the epidermis of aquatic species (goldfish and newt) where a stratum corneum is absent or very thin. The molecular weight of transglutaminase immunoreactive bands generally varies between 40 to 62 kDa, with the most represented bands at 52-57 kDa in most species. The more intense loricrin- and sciellin-immunoreactive bands are seen at 50-55-62 kDa, but are weak or absent in aquatic vertebrates. Loricrine-like immunoreactivity is present in the epidermis where alpha-(soft)-keratinization occurs. Isopeptide bonds are mainly associated to bands in the range of 50-62 kDa. In vertebrates where hard-keratin is expressed (the beta-keratin corneous layer of sauropsids and in feathers) or in hair cortex of mammals, no loricrin-like, transglutaminase-, and isopeptide-bond-immunoreactivities are seen. Immunoblotting however shows loricrin-, sciellin-, and trasnsglutaminase-positive bands in the corneous layers containing beta-keratin. Histologically, the epidermis of most amniotes shows variable transglutaminase immunoreactivity, but isopeptide-bond and sciellin immunoreactivities are weak or undetactable in most species. The limitations of immunohistochemical methods are discussed and compared with results from immunoblotting. In reptilian epidermis transglutaminase is mainly localized in 0.15-0.3 microm dense granules or diffuse in transitional alpha-keratogenic cells. In beta-keratogenic cells few small dense granules show a weak immunolabeling. Transglutaminase is present in nuclei of terminal differentiating alpha- and beta-keratinocytes, as in those of mature inner and outer root sheath. The present study suggests that keratinization based on loricrin, sciellin and transglutaminase was probably present in the stratum corneoum of basic amniotes in the Carboniferous. These proteins were mainly maintained in alpha-keratogenic layers of amniotes but decreased in beta-keratogenic layers of sauropsids (reptiles and birds). The study suggests that similar proteins for the formation of the cornified cell envelope are present in alpha-keratinocytes across vertebrates but not in beta-keratinocytes.     

Keratinization in the epidermis of amphibians and the lungfish: comparison with amniote keratinization

Keratinization in the epidermis of amphibians and the lungfish has been studied by electron microscopy, autoradiography and immunocytochemistry to determine whether histidine-rich proteins, filaggrin and loricrin are present. In the lungfish and amphibian tadpoles, anti-keratin antibodies (AE1 and AE3) stain the whole epidermis but not the AE2 antibody, a marker for keratinization. In adult epidermis, the AE2 antibody mainly stains keratinized layers, AE1 mainly stained basal cells, less suprabasal cells and no pre-keratinized and keratinized layers, and AE3 stains all epidermal layers. This staining pattern resembles that of amniote epidermis. Little tritiated histidine is taken up in toad epidermis at 4-6 h post-injection but 24 h after injection the radioactivity is most concentrated in the replacement layer beneath the corneus. This indicates that protein synthesis takes place in the epidermis but, due to the metabolic conversion that takes place in 24 h, it is unlikely that histidine-rich proteins are formed. Neither filaggrin-like nor loricrine-like immunoreactivities are present in amphibian and lungfish epidermis. This indicates absence of histidine-rich matrix proteins and corneous cell envelope proteins and only mucus is present among keratin filaments. Filaggrine-like and loricrin-like proteins are characteristic of amniotes epidermis and might have originated in basic amniotes (cotylosaurs).     

Ultrastructural localization of alpha-keratins in the regenerating epidermis of the lizard Podarcis muralis during formation of the shedding layer

In the epidermis of lizards, alpha- and beta-keratins are sequentially produced during a shedding cycle. Using pre- and post-embedding immunocytochemistry this study shows the ultrastructural distribution of 3 alpha-keratin antibodies (AE1, AE2, AE3) in the renewing epidermis and in the shedding complex of the regenerating tail of the lizard Podarcis muralis. The AE1 antibody that recognizes acidic low MW keratins is confined to tonofilament bundles in basal and suprabasal cells but is not present in keratinizing beta- and alpha-cells. The AE2 antibody that recognises higher MW keratins weakly stains pre-keratinized cells and intensely keratinized alpha-layers. A weak labeling is present in small electrondense areas within the beta-layer. The AE3 antibody, that recognizes low and high MW basic keratins, immunolabels tonofilament bundles in all epidermal layers but intensely the alpha-keratinizing and keratinized layers (mesos, alpha-, lacunar and clear). Keratohyalin-like granules, present in the clear cells of the shedding layer, are negative to these antibodies so that the cornified clear layer contains keratins mixed with non-keratin material. The AE3 antibody shows that the mature beta-layer and the spinulated folds of the oberhautchen are labeled only in small dense areas among the prevalent electron-pale beta-keratin material. Therefore, some alpha-keratin is still present in the beta-layer, and supports the idea that alpha-keratins (basic) function as scaffold for beta-keratin deposition.     

Immunological characterization and fine localization of a lizard beta-keratin

Scales of lizards contain beta-keratin of poorly known composition. In the present study, a rat polyclonal serum against a lizard beta-keratin of 14-15 kDa has been produced and the relative protein has been immunolocalized in the epidermis. The observations for the first time show that the isolated protein band derives from the extraction of a protein component of the beta-keratin filaments of lizard epidermis. In immunoblots and immunocytochemistry, the antiserum recognizes most lizard beta-keratins, but produces a variable cross-reactivity with snake beta-keratins, and weak or no reactivity with beta-keratins isolated from tuatara, turtles, alligator and birds. In bidimensional immunoblots of lizard epidermis, three main spots at 15-16 kDa with isoelectric point at 7.0, 7.6 and 8.0, and an unresolved large spot at 29-30 kDa and with pI at 7.5-8.0, are obtained, may be derived from the aggregation of smaller beta-keratin proteins. The ultrastructural immunolocalization with the antibody against lizard beta-keratin shows that only small and large beta-keratin filaments of beta-cells of lizard epidermis are labeled. Keratin bundles in oberhautchen cells are less immunolabeled. Beta-keratin is rapidly polymerized into beta-packets that merge into larger beta-keratin filaments. No labeling is present over other cell organelles or cell layers of lizard epidermis, and is absent in non-epidermal cells. The antiserum recognizes epitope(s) characteristics for lizard beta-keratins, partially recognized in snakes and absent in non-lepidosaurian species. This result indicates that beta-keratins among different reptilian groups posses different immunoreactive regions.     

Immunocytochemical observations on the cornification of soft and hard epidermis in the turtle Chrysemys picta

The process of cornification in the shell and non-shelled areas of the epidermis of the turtle Chrysemys picta was analyzed by light and ultrastructural immunohistochemistry for keratins, filaggrin and loricrin. Beta-keratin (hard keratin) was only present in the corneus layer of the plastron and carapace. The use of a beta-keratin antibody, developed against a specific chick scale beta-keratin, demonstrated that avian and reptilian hard keratins share common amino acid sequences. In both, shelled and non-shelled epidermis, acidic alpha keratin (AE1 positive) was limited to tonofilament bundles of the basal and suprabasal layer, while basic keratin (AE3 positive) was present in basal, suprabasal, and less intensely, pre-corneus layers, but tended to disappear in the corneus layer. The AE2 antibody, which in mammalian epidermis recognizes specific keratins of cornification, did not stain turtle shell but only the corneus layer of non-shelled (soft) epidermis. Two and four hours after an injection of tritiated histidine, the labelling was evenly distributed over the whole epidermis of both shelled and non-shelled areas, but was absent from the stratum corneum. In the areas of growth at the margin of the scutes of the shell, the labelling increased in precorneus layers. This suggests that histidine uptake is only related to shell growth and not to the production of a histidine-rich protein involved in keratinization. No filaggrin-like and loricrin-like immunoreactivity was seen in the carapace or plastron epidermis. However, in both proteins, some immunoreactivity was found in the transitional layer and in the lower level of the corneus layer of non-shelled areas. Loricrin- and filaggrin-like labelling was seen in small organelles (0.05-0.3 mum) among keratin bundles, identified with mucous-like granules and vesicular bodies. These organelles, present only in non-shelled epidermis, were more frequent along the border with the corneus layer, and labelling was low to absent in mature keratinocytes. This may be due to epitope masking or degradation. The immunolabelling for filaggrin was seen instead in the extracellular space among mature keratinocytes, over a material previously identified as mucus. The possibility that this labelling identified some epitopes derived from degraded portions of a filaggrin-like molecule is discussed. The present study suggests that proteins with some filaggrin- and loricrin-immunoreactivity are present in alpha-keratinocytes but not in beta-keratin cells of the shell.     

Cornification in reptilian epidermis occurs through the deposition of keratin‐associated beta‐proteins (beta‐keratins) onto a scaffold of intermediate filament keratins

The isolation of genes for alpha‐keratins and keratin‐associated beta‐proteins (formerly beta‐keratins) has allowed the production of epitope‐specific antibodies for localizing these proteins during the process of cornification epidermis of reptilian sauropsids. The antibodies are directed toward proteins in the alpha‐keratin range (40–70 kDa) or beta‐protein range (10–30 kDa) of most reptilian sauropsids. The ultrastructural immunogold study shows the localization of acidic alpha‐proteins in suprabasal and precorneous epidermal layers in lizard, snake, tuatara, crocodile, and turtle while keratin‐associated beta‐proteins are localized in precorneous and corneous layers. This late activation of the synthesis of keratin‐associated beta‐proteins is typical for keratin‐associated and corneous proteins in mammalian epidermis (involucrin, filaggrin, loricrin) or hair (tyrosine‐rich or sulfur‐rich proteins). In turtles and crocodilians epidermis, keratin‐associated beta‐proteins are synthesized in upper spinosus and precorneous layers and accumulate in the corneous layer. The complex stratification of lepidosaurian epidermis derives from the deposition of specific glycine‐rich versus cysteine‐glycine‐rich keratin‐associated beta‐proteins in cells sequentially produced from the basal layer and not from the alternation of beta‐ with alpha‐keratins. The process gives rise to Oberhäutchen, beta‐, mesos‐, and alpha‐layers during the shedding cycle of lizards and snakes. Differently from fish, amphibian, and mammalian keratin‐associated proteins (KAPs) of the epidermis, the keratin‐associated beta‐proteins of sauropsids are capable to form filaments of 3–4 nm which give rise to an X‐ray beta‐pattern as a consequence of the presence of a beta‐pleated central region of high homology, which seems to be absent in KAPs of the other vertebrates. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

Immunocytochemical and autoradiographic studies on the process of keratinization in avian epidermis suggests absence of keratohyalin

The process of keratinization in apteric avian epidermis and in scutate scales of some avian species has been studied by autoradiography for histidine and immunohistochemistry for keratins and other epidermal proteins. Acidic or basic alpha-keratins are present in basal, spinosus, and transitional layers, but are not seen in the corneous layer. Keratinization-specific alpha-keratins (AE2-positive) are observed in the corneous layer of apteric epidermis but not in that of scutate scales, which contain mainly beta-keratin. Alpha-keratin bundles accumulate along the plasma membrane of transitional cells of apteric epidermis. In contrast to the situation in scutate scales, in the transitional layer and in the lowermost part of the corneous layer of apteric epidermis, filaggrin-like, loricrin-like, and transglutaminase immunoreactivities are present. The lack of isopeptide bond immunoreactivity suggests that undetectable isopeptide bonds are present in avian keratinocytes. Using immunogold ultrastructural immunocytochemistry a low but localized loricrin-like and, less, filaggrin-like labeling is seen over round-oval granules or vesicles among keratin bundles of upper spinosus and transitional keratinocytes of apteric epidermis. Filaggrin-and loricrin-labeling are absent in alpha-keratin bundles localized along the plasma membrane and in the corneous layer, formerly considered keratohyalin. Using ultrastructural autoradiography for tritiated histidine, occasional trace grains are seen among these alpha-keratin bundles. A different mechanism of redistribution of matrix and corneous cell envelope proteins probably operates in avian keratinocytes as compared to that of mammals. Keratin bundles are compacted around the lipid-core of apteric epidermis keratinocytes, which do not form complex chemico/mechanical-resistant corneous cell envelopes as in mammalian keratinocytes. These observations suggest that low amounts of matrix proteins are present among keratin bundles of avian keratinocytes and that keratohyalin granules are absent.     

Comparative immunolocalization of keratin‐associated beta‐proteins (beta‐keratins) supports a new explanation for the cyclical process of keratinocyte differentiation in lizard epidermis

Lizard epidermis is made of beta‐ and alpha‐layers. Using Western blot tested antibodies, the ultrastructural immunolocalization of specific keratin‐associated beta‐proteins in the epidermis of different lizard species reveals that glycine‐rich beta‐proteins (HgG5) localize in the beta‐layer, while glycine–cysteine‐medium‐rich beta‐proteins (HgGC10) are present in oberhautchen and alpha‐layers. This suggests a new explanation for the formation of different epidermal layers during the shedding cycle in lepidosaurian epidermis instead of an alternance between beta‐keratins and alpha‐keratins. It is proposed that different sets of genes coding for specific beta‐proteins are activated in keratinocytes during the renewal phase of the shedding cycle. Initially, glycine–cysteine‐medium‐rich beta‐proteins with hydrophilic and elastic properties accumulate over alpha‐keratins in the oberhautchen but are replaced in the next cell layer with glycine‐rich hydrophobic beta‐proteins forming a resistant, stiff, and hydrophobic beta‐layer. The synthesis of glycine‐rich proteins terminates in mesos and alpha‐cells where these proteins are replaced with glycine–cysteine‐rich beta‐proteins. The pattern of beta‐protein deposition onto a scaffold of intermediate filament keratins is typical for keratin‐associated proteins and the association between alpha‐keratins and specific keratin‐associated beta‐proteins during the renewal phase of the shedding cycle gives rise to epidermal layers possessing different structural, mechanical, and texture properties.     

Loricrin-like immunoreactivity during keratinization in lizard epidermis

Two modalities of keratinization are present in lizard epidermis: alpha (soft-pliable corneous layers) and beta (hard and inflexible corneous layers). While beta-keratinization is probably due to the synthesis of a new (beta)-keratin gene product, alpha keratinization resembles in part that of mammalian epidermis. The goal of this study was to test whether a sulfur-rich molecule similar to the mammalian corneous cell envelope protein loricrin is also present in lizard epidermis. This was done using X-ray microanalysis and immunocytochemical and ultrastructural methods. In the epidermis of the lizard Podarcis muralis small (0.1-0.3 microm) to large (1-5 microm) keratohyalin-like granules (KHLGs) are produced in alpha-keratinizing cells, especially in the clear layer. Small KHLGs contain sulfur and show weak filaggrin-like and stronger loricrin-like immunoreactivities. The latter is also present in keratinizing alpha-layers but is absent in the beta layers. Large KHLGs in the clear layer derive from the aggregation of the small granules with other components, including lipid material. These large granules show some loricrin-like immunoreactivity and contain sulfur and phosphorous, histidine, but not filaggrin-like immunoreactivity. It is suggested here that phosphorous derives from their phospholipid component. The present study shows that the modality of alpha-keratinization of lizard epidermis resembles that of mammals and suggests that the basic molecular mechanisms of keratin aggregation and formation of the corneous cell envelope were already present in the therapsid line of reptiles from which mammals evolved.     

Immunogold labeling shows that glycine‐cysteine‐rich beta‐proteins are deposited in the Oberhäutchen layer of snake epidermis in preparation to shedding

Shedding in snakes is cyclical and derives from the differentiation of an intraepidermal shedding complex made of two different layers, termed clear and Oberhäutchen that determine the separation between the outer from the inner epidermal generation that produces a molt. The present comparative immunocytochemical study on the epidermis and molts of different species of snakes shows that a glycine‐cysteine‐rich corneous beta‐protein in a snake is prevalently accumulated in cells of the Oberhäutchen layer and decreases in those of the beta‐layer. The protein is variably distributed in the mature beta‐layer of species representing some snake families when the beta‐layer merges with the Oberhäutchen but disappears in alpha‐layers. Therefore, this protein represents an early marker of the transition between the outer and the inner epidermal generations in the epidermis of snakes in general. It is hypothesized that specific gene activation for glycine‐cysteine‐rich corneous beta‐proteins occurs during the passage from the clear layer of the outer epidermal generation to the Oberhäutchen layer of the replacing inner epidermal generation. It is suggested that in the epidermis of most species glycine‐cysteine‐rich corneous beta‐proteins form part of the dense corneous material that rapidly accumulates in the differentiating Oberhäutchen cells but decreases in the following beta‐layer of the inner epidermal generation destined to be separated from the previous outer generation in the process of shedding. The regulation of the synthesis of these and other proteins is, therefore, crucial in timing the different stages of the shedding cycle in lepidosaurian reptiles. J. Morphol. 276:144–151, 2015. © 2014 Wiley Periodicals, Inc.     

Presence of putative histidine-rich proteins in the amphibian epidermis

In amphibian epidermis mucus is thought to constitute the matrix material that links keratin filaments present in cells of the corneous layer. As contrast in mammals, and perhaps in all amniotes, histidine-rich proteins form the matrix material. In order to address the study of matrix molecules in the epidermis of the first tetrapods, the amphibians, an autoradiographic and electrophoretic study has been done after administration of tritiated histidine. Histological analysis of amphibian epidermis shows that histidine is taken up in the upper intermediate and replacement layers beneath the corneous layer. Ultrastructural autoradiographic analysis reveals that electron-dense interkeratin material is labeled after administration of tritiated histidine. Electrophoretic analysis of the epidermis shows labeled proteic bands at 58-61, 50-55, 40-45, and some only weakly labeled at 30 and 24-25 kDa at 4-48 hours after injection of tritiated histidine. Keratin markers show that bands at 40-61 kDa contain keratins. Most histidine is probably converted into other amino acids such as glutamate and glutamine that are incorporated into newly synthetized keratins. However, non-keratin histidine-incorporating proteins within the keratin range could also be formed. The bands at 30 and 24-25 kDa suggest that these putative histidine-rich proteins are not keratins. In fact, their molecular weigh is below the range of that for keratins. In contrast with the mammalian condition, but resembling reports for lizard epidermis, putative histidine-rich proteins in amphibians have no high molecular weight precursor. Although filaggrin is not detectable by immunofluorescence in sections of amphibian epidermis, protein extraction, electrophoresis and immunoblotting are more sensitive. In the epidermis of toad and frog, but only occasionally in that of newt, filaggrin cross-reactive proteic bands are seen at 50-55, 40-45, and sometimes at 25 kDa. This suggests that after extraction and unmasking of reactive sites in the epidermis of more terrestrial amphians (anurans), some HRPs with filaggrin-like cross-reactivity are present. The overlap that exists at 50-55 kDa between filaggrin-positive and AE2-positive keratins, but not that at 40-45 kDa further indicate that non-keratin, filaggrin-like proteins may be present in anuran epidermis. The present study suggests for the first time that very small amounts of histidine-rich proteins are produced among keratin filaments in upper intermediate, replacement and corneous layers of amphibian epidermis. Although the molecular composition of these proteins is unknown, precluding understanding of their relationship to those of mammals and reptiles, these cationic proteins might have originated in conjunction with the formation of a horny layer during the adaptation to land during the Carboniferous and were possibly refined later in the epidermis of amniotes.     

Keratinization and lipogenesis in epidermal derivatives of the zebrafinch, Taeniopygia guttata castanotis (Aves, Passeriformes, Ploecidae) during embryonic development

Little is known of the lipid content of beta-keratin-producing cells such as those of feathers, scutate scales, and beak. The sequence of epidermal layers in some apteria and in interfollicular epidermis in the zebrafinch embryo (Taeniopygia guttata castanotis) was studied. Also, the production of beta-keratin in natal down feathers and beak was ultrastructurally analyzed in embryos from 3-4 to 17-18 days postdeposition, before hatching. Two layers of periderm initially cover the embryo, but there are eventually 6-8 over the epidermis of the beak. In the beak and sheath cells of feathers, peridermal granules are numerous at 12-14 days postdeposition but they are less frequent in apteria. These granules swell and disappear during sheath or peridermal degeneration at 15-17 days postdeposition. A thin beta-keratin layer forms under the periderm among feather germs of pterylous areas but is discontinuous or disappears in apteria. In differentiating cells of barbs, barbules, and calamus cells of natal down, electron-dense beta-keratin filaments form bundles oriented along the main axis of these cells. Cells of the pulp epidermis and collar, at the base of the follicle, contain lipids and bundles of alpha-keratin filaments. Degenerating pulp cells show vacuolization and nuclear pycnosis. During beta-keratin packing, keratin bundles turn electron-pale, perhaps due to the addition of lipids to produce the final, homogenous beta-keratin matrix. In contrast to the situation in feathers, in the cells of beak beta-keratin packets are irregularly oriented. In both feather and beak epidermal cells the Golgi apparatus and smooth endoplasmic reticulum produce vesicles containing lipid-like material which is also found among forming beta-keratin. The contribution of lipids or lipoprotein to the initial aggregation of beta-keratin molecules is discussed.     

Characterization of beta-keratins and associated proteins in adult and regenerating epidermis of lizards

Reptilian epidermis contains two types of keratin, soft (alpha) and hard (beta). The biosynthesis and molecular weight of beta-keratin during differentiation of lizard epidermis have been studied by autoradiography, immunocytochemistry and immunoblotting. Tritiated proline is mainly incorporated into differentiating and maturing beta-keratin cells with a pattern similar to that observed after immunostaining with a chicken beta-keratin antibody. While the antibody labels a mature form of beta-keratin incorporated in large filaments, the autoradiographic analysis shows that beta-keratin is produced within the first 30 min in ribosomes, and is later packed into large filaments. Also the dermis incorporates high amount of proline for the synthesis of collagen. The skin was separated into epidermis and dermis, which were analyzed separately by protein extraction and electrophoresis. In the epidermal extract proline-labeled proteic bands at 10, 15, 18-20, 42-45, 52-56, 85-90 and 120 kDa appear at 1, 3 and 5 h post-injection. The comparison with the dermal extract shows only the 85-90 and 120 kDa bands, which correspond to collagen. Probably the glycine-rich sequences of collagen present also in beta-keratins are weakly recognized by the beta-1 antibody. Immunoblotting with the beta-keratin antibody identifies proteic bands according to the isolation method. After-saline or urea-thiol extraction bands at 10-15, 18-20, 40, 55 and 62 kDa appear. After extraction and carboxymethylation, weak bands at 10-15, 18-20 and 30-32 kDa are present in some preparations, while in others also bands at 55 and 62 kDa are present. It appears that the lowermost bands at 10-20 kDa are simple beta-keratins, while those at 42-56 kDa are complex or polymeric forms of beta-keratins. The smallest beta-keratins (10-20 kDa) may be early synthesized proteins that are polymerized into larger beta-keratins which are then packed to form larger filaments. Some proline-labeled bands differ from those produced after injection of tritiated histidine. The latter treatment does not show 10-20 kDa labeled proteins, but tends to show bands at 27, 30-33, 40-42 and 50-62 kDa. Histidine-labeled proteins mainly localize in keratohyalin-like granules and dark keratin bundles of clear-oberhautchen layers of lizard epidermis, and their composition is probably different from that of beta-keratin.